Folates are essential for life and folate deficiency contributes to a range of health problems including cardiovascular disease, fetal abnormalities, neurologic disorders, and cancer. Antifolates, represented by methotrexate and, more recently, raltitrexed and pemetrexed, continue to occupy a unique and important niche among the modern day pharmacopoeia for cancer along with other pathologic conditions. This renewal application for a grant currently in its 19th year proposes to better understand the biology and therapeutic potential of the human reduced folate carrier (hRFC) and human proton-coupled folate transporter (hPCFT), two of the major membrane transport systems for folates and antifolates in human cells, tissues and tumors which are widely expressed, yet are functionally and anatomically distinct. Our structure-function studies of hRFC have provided substantial insights into its membrane topology, N-glycosylation, important domains and amino acids, and three-dimensional packing associations. Most recently, we began to characterize hRFC quaternary structure and unambiguously identified hRFC homo-oligomers composed of monomeric hRFC molecules, and obtained compelling evidence for a negative dominance of wild type hRFC by inactive hRFC mutants, involving downstream effects on intracellular trafficking and/or functional coupling between hRFC monomers. Following identification of the low pH folate transporter, hPCFT, in 2006, we began a series of studies designed to directly assess the possible therapeutic importance of this transporter vis ` vis hRFC for cancer. Key reagents were generated including stably transfected cell lines (including Tet-regulated models), a functional cysteine-less hPCFT construct, antibodies to the hPCFT protein, and, perhaps most exciting, the first generation of specific and potent hPCFT cytotoxic substrates that are not transported by hRFC. Aim 1 of our proposed study will characterize the structural, functional, and regulatory features of hRFC homo- oligomers. A wide range of biochemical, cellular, and molecular approaches will be used to identify the minimum functional unit and characterize the structure of oligomeric hRFC, and to explore the implications of hRFC oligomers to transport mechanism, antifolate resistance, and biochemical modulation. Aim 2 will establish the therapeutic potential, along with the structural and functional features of hPCFT. The focus is overtly on possibilities for therapeutic targeting tumors with cytotoxic antifolate drugs that are selective for hPCFT (and folate receptors) but are not transported by hRFC. Proposed studies include assays of hPCFT and hRFC tumor expression and activity profiles, functional stoichiometries for hRFC and hPCFT, and the therapeutic impact of concomitant tumor expression of these transport systems, as well as folate receptors. Our proposed studies are strongly supported by our preliminary and published results, and are distinctive for their novelty and likelihood of providing critical new insights into mechanism and regulation of these physiologically and pharmacologically important membrane transporters. PUBLIC HEALTH RELEVANCE: Folates are essential for life and folate deficiency contributes to a range of health problems including cardiovascular disease, fetal abnormalities, neurologic disorders, and cancer. Antifolates, represented by methotrexate and, more recently, raltitrexed and pemetrexed, continue to occupy a unique and important niche among the modern day pharmacopoeia for cancer along with other pathologic conditions. The application proposes to better understand the biology and therapeutic potential of the human reduced folate carrier (hRFC) and human proton-coupled folate transporter (hPCFT), two of the major membrane transport systems for folates and antifolates in human cells, tissues and tumors which are widely expressed, yet are functionally and anatomically distinct. Our proposed studies provide an important prelude for the design of new therapeutics for cancer including solid tumors and approaches for therapeutically altering transporter levels and function in the context of dietary folate supplementation and antifolate therapy of cancer and other diseases.